Open Access Dissertation
Date of Award
Mary Kay H. Pflum
Post-translational modifications (PTMs) are responsible for a variety of cellular processes. One such PTM is protein phosphorylation, which is catalyzed by kinases. Kinase enzymes play important roles in cellular signaling pathways, but dysregulation of kinase-mediated events results in the formation of diseases, which make kinases favorable drug targets. To uncover the role kinases play in the development of diseases, kinase-mediated cellular events need to be better understood. The current gap in the field is the lack of tools available to identify the kinase that is responsible for specific phosphorylation events within the cell. To improve the gap in the field, the Pflum lab has developed kinase-catalyzed labeling and crosslinking techniques used to identify kinase-substrate pairs. The techniques utilize γ-modified ATP analogs that contain tags or crosslinking motifs which help identify kinase-substrate discovery by labeling substrates or stabilizing the kinase-substrate interactions through crosslinking. The K-CLIP (kinase-catalyzed crosslinking and immunoprecipitation) method was applied to additional human lysates, as well as yeast and tissue homogenates for the first time. K-CLIP was successfully applied to two collaborative studies, which helped uncover unknown kinase-mediated cell biology. Next, additional affinity-based crosslinking ATP analogs, ATP-aryl fluorosulfate (ATP-AFS) and ATP-hexanoyl bromide (ATP-HexBr), were developed to avoid UV light and undergo crosslinking with other nucleophilic amino acids. The new analogs were used to show the electrophiles were able to react with more than Cys amino acids, which was demonstrated with PKA, EGFR, and SRC kinases. The ATP-AFS analog was found to more robustly form covalent crosslinks and hence, was utilized to demonstrate kinase-substrate pair covalent crosslinking and crosslinking in a complex lysate system. Finally, the K-BILDS (kinase-catalyzed biotinylation with inactivated lysates for substrate discovery) method was modified to use for a collaborative study and to develop a consensus site identification tool. We successfully implemented K-BILDS in a highly purified FACS (focal adhesion cytoskeleton) complexes to identify unknown substrates of PKA related to cell motility. Also, the K-BILDS method was redeveloped to use as a consensus site identification tool, which is still under development, but will provide an additional method to help uncover consensus sequences for all 535 kinases. In summary, various tools were utilized and developed to help discover kinase-substrate pairs and uncover unknown kinase-mediated cell biology, which is an essential need in the kinase field.
Beltman, Rachel, "Kinase-Catalyzed Labeling To Identify Kinase-Substrate Pairs Using Γ-Phosphate Modified Atp Analogs" (2022). Wayne State University Dissertations. 3660.